Multi-color Molecular Visualization of Signaling Proteins Reveals How C-Terminal Src Kinase Nanoclusters Regulate T Cell Receptor Activation.

Elucidating the mechanisms that controlled T cell activation requires visualization of the spatial organization of multiple proteins on the submicron scale. Here, we use stoichiometrically accurate, multiplexed, single-molecule super-resolution microscopy (DNA-PAINT) to image the nanoscale spatial architecture of the primary inhibitor of the T cell signaling pathway, Csk, and two binding partners implicated in its membrane association, PAG and TRAF3. Combined with a newly developed co-clustering analysis framework, we find that Csk forms nanoscale clusters proximal to the plasma membrane that are lost post-stimulation and are re-recruited at later time points.

Crispr/Cas Mediated Deletion of PTPN22 in Jurkat T Cells Enhances TCR Signaling and Production of IL-2.

A single nucleotide polymorphism, C1858T, in the gene encoding the protein tyrosine phosphatase nonreceptor type 22 () results in one of the strongest genetic traits associated with autoimmune disease outside of the Major Histocompatibility Complex (MHC) genes. However, the consequences of this polymorphism, which introduces an arginine to tryptophan substitution at amino acid 620, for the function of PTPN22 protein is unclear and conflicting results have been obtained in human compared to mouse cells expressing this variant phosphatase. In mouse the variant appears to be a loss-of-function allele resembling a milder form of the null allele, while studies in human cells have reported it to be a gain-of-function mutation.